New approaches for the control and eradication of schistosomiasis in Venezuela

Schistosomiasis in Americawith the exception of Brazil, behaves as a chronic mild disease with few clinical manifestations due to low parasite burden. These features restrict the clinical and parasitological diagnosis. The most commonly used stool examination method, Kato-Katz, becomes intensitive when the majority of individuals excrete less than 100 eggs/g of feces. In view that antigen-detecting techniques have not been able to reveal light infections, the antibody detecting assays remain as a very valuable diagnostic tool for epidemiological surveillance. The Venezuelan Schistosomiasis Research group (CECOICE) has designed a mass chemotherapy strategy based on sero-diagnosis. Since blood sampling is one of the important limitating factors for large seroepidemiological trials we developed a simple capillary technique that sucessfully overcomed most of the limitations of blood drawing. In this sense, ELISA seems to be the most adecuate test for epidemiological studies. Soluble egg Schistosoma mansoni antigen (SEA) has been largely used in Venezuela. The sensitivity ELISA-SEA in our hands is 90% moreover its specific reach 92% when populations from non-endemic areas but heavily infected with other intestinal parasites are analyzed. The Schistosomiasis Control Program is currently carrying out the surveillance of endemic areas using ELISA-SEA as the first screening method, followed by the Circumoval Precipitin test for validation assay. The results with these two serological techniques allowed us to defined the criteria of chemotherapy in populations of the endemic areas. On the search of better diagnostic technique, Alkaline Phosphatase Immunoenzyme Assay (APIA) is being evaluated in field surveys.

Blastocystis hominis: occurrence in children and staff members of municipal day-care centers from Botucatu, São Paulo State, Brazil

To study the frequency of Blastocystis hominis among healthy individuals, feces were collected from 153 children and 20 staff members of some municipal day-care centers. Three separate stool specimens of each individual were processed by Lutz and Faust methods. From 173 studied individuals, 60 (34.7%) showed B. hominis, frequently in association with other intestinal parasites and/or commensals. B. hominis was found mainly in adults and children between 36 and 72 months old. All positive cases were detected only by Lutz method and the use of three stool specimens increased the positivity of the parasitological diagnostic.

Cryptosporidium sp. in children suffering from acute diarrhea at Uberlândia City, State of Minas Gerais, Brazil

This study's objective was to search for Cryptosporidium sp. in diarrheic feces from children aged zero to 12 years and cared for at medical units within Universidade Federal de Uberlândia or at a private practice in Uberlândia, State of Minas Gerais, Brazil, from September 1992 to August 1993. Three fecal samples preserved in 10% formalin, were collected from 94 children. Oocyst concentration was performed through Ritchie's (modified) method and staining of fecal smears for each sample (total of 1128 slides) was done by the "Safranin/Methylene Blue" and the "Kinyoun (modified)" techniques. The Hoffmann, Pons & Janer method was also employed to look for other enteroparasites. From 94 children, 4.26% excreted fecal Cryptosporidium oocysts. The infection seemed to vary according to age: 5.08% of patients aged zero to two years old; 33.33% of those aging eight to ten years (P>0.05). Cryptosporidium appeared in November, December and March, during the rainy season. 20.21% of the children harbored at least one enteroparasite different from Cryptosporidium, mainly Giardia intestinalis (12.77%). From Cryptosporidium infected patients, two had only this kind, another harbored Giardia intestinalis; the last one hosted Strongyloides stercoralis.

Strongyloides stercoralis and other Enteroparasites in Children at Uberlândia City, State of Minas Gerais, Brazil

To evaluate the rate of infection by Strongyloides stercoralis and other enteroparasites a survey was conducted in the city of Uberlândia, State of Minas Gerais, Brazil. A total of 900 stool samples from 300 children aging from four months to seven years, randomly selected in ten nursery schools from September 1994 to December 1995, were examined, both by the Baermann-Moraes and Lutz methods. Thirty nine children (13%) were found to be infected by S. stercoralis, 64.1% were boys and 35.9% were girls. Taking all the enteroparasites as a whole the results of the survey pointed out that 265 (88.4%) of the 300 children were infected by the following: Giardia lamblia, 78.3%; Ascaris lumbricoides, 15.3%; S. stercoralis, 13%; Hymenolepis nana, 6.7%; hookworms, 6%; Enterobius vermicularis, 4%; Hymenole-pis diminuta, 4% and Trichuris trichiura, 0.7%. From 265 infected children 64.5% were mono-infected, 27.2% were infected by two parasites and 8.3% had a poly-specific parasite burden. It was concluded that strongyloidiasis is hyperendemic in this area

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37°C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.

Parasite remains in archaeological sites

Organic remains can be found in many different environments. They are the most significant source for paleoparasitological studies as well as for other paleoecological reconstruction. Preserved paleoparasitological remains are found from the driest to the moistest conditions. They help us to understand past and present diseases and therefore contribute to understanding the evolution of present human sociality, biology, and behavior. In this paper, the scope of the surviving evidence will be briefly surveyed, and the great variety of ways it has been preserved in different environments will be discussed. This is done to develop to the most appropriated techniques to recover remaining parasites. Different techniques applied to the study of paleoparasitological remains, preserved in different environments, are presented. The most common materials used to analyze prehistoric human groups are reviewed, and their potential for reconstructing ancient environment and disease are emphasized. This paper also urges increased cooperation among archaeologists, paleontologists, and paleoparasitologists.

Strongyloides ratti antigenic components recognized by IgE antibodies in immunoblotting as an additional tool for improving the immunodiagnosis in human strongyloidiasis

IgE antibody response in human strongyloidiasis was evaluated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) using Strongyloides ratti saline extract as heterologous antigen. A total of 50 serum samples of patients who were shedding S. stercoralis larvae in feces (group I, copropositive), 38 of patients with other intestinal parasites (group II), and 38 of subjects with negative results in three parasitologic assays (group III, copronegative) were analyzed. Levels of IgE anti-Strongyloides expressed in ELISA Index (EI) were significantly higher in patients of group I (1.32) than in group II (0.51) and group III (0.81), with positivity rates of 54%, 0%, and 10.5%, respectively. Fifteen S. ratti antigenic components were recognized in IB-IgE by sera of group I, with frequency ranging from 8% to 46%. In group II, only two antigenic bands (101, 81 kDa) were detected in a frequency of 10% and no reactivity was found in group III. Sera with EI values > 1.5 recognized five from 13 specific antigenic bands (70, 63, 61, 44, 7 kDa). It can be concluded that these five antigenic components recognized by IB-IgE using S. ratti antigen might be employed as an additional tool for improving the immunodiagnosis in human strongyloidiasis.

Related factors to human toxocariasis in a rural community of Argentina

The objective of this study was to evaluate the relationship between toxocariasis frequency and demographic, environmental, sanitary variables, eosinophylia, and other intestinal parasites in a rural population of Argentina. Serological examination of 100 individuals was carried out by using ELISA technique for the detection of anti-toxocara antibodies. Eosinophiles in peripheral blood, presence of intestinal parasites, and demographic, environmental, and socio-cultural data were evaluated. Eighty-one feces samples of dogs belonging to the studied people were analyzed to detect eggs of Toxocara canis. Thirty of them were from 30 dogs and 51 were pools from dog feces. Samples of dirt from around the homes (n: 47) and from public park (n: 4) were taken. To determine the associations, the c² and Fisher tests were used. The seroprevalence was 23%. Eosinophilia in peripheral blood was detected in 86.95% seropositive individuals and in 37.66% seronegative individuals (p < 0.001, OR = 11.03). Of the 23 people with positive serology, 69.56% had at least one intestinal parasite. All individuals with positive serology had dogs in their homes. Among the dog owners there was a significant association between the presence of anti-toxocara antibodies and home flooding. Eggs of T. canis were detected in the feces of 5/81 dogs and three of these dogs belonged to individuals with positive serology. Eggs of Toxocara spp. were found in 41.17% of the dirt samples, eight of which came from the area surrounding the homes of individuals with positive serology (p = 0.032; OR = 4.36). Taking into account all the variables influencing the frequency of toxocariasis in this population, the implementation of Public Health programs specifically focused on anti-parasitic treatment of dogs is recommended.

Low transmission areas of schistosomiasis in Venezuela: consequences on the diagnosis, treatment, and control

Schistosomiasis low transmission areas as Venezuela, can be defined as those where the vector exists, the prevalence of active cases is under 25%, individuals with mild intensity of infection predominate and are mostly asymptomatic. These areas are the consequence of effective control programs, however, "silent" epidemiological places are difficult to trace, avoiding the opportune diagnosis and treatment of infected persons. Clinic and abdominal ultrasound have not shown to discriminate infected from uninfected persons in areas where besides Schistosoma mansoni, intestinal parasites are the rule. Under these conditions, serology remains as a very valuable diagnostic tool, since it gives a closer approximation to the true prevalence. In this sense, circumoval precipitin test, ELISA-SEA with sodium metaperiodate, and alkaline phosphatase immunoassay joined to coprology allow the identification of the "schistosomiasis cases". In relation to public health, schistosomiasis has been underestimated by the sanitary authorities and the investment on its control is being transferred to other diseases of major social and political relevance neglecting sanitary efforts and allowing growth of snail population. Some strategies of diagnosis and control should be done before schistosomiasis reemergence occurs in low transmission areas.

Parasitism in Kansas in the 1800s: a glimpse to the past through the analysis of grave sediments from Meadowlark cemetery

During the excavations of the XIX century Meadowlark cemetery (Manhattan, Kansas, US), samples of sediments were taken from around five skeletons, and analyzed to detect intestinal parasites. No helminth eggs were found, but immunological ELISA tests for Entamoeba histolytica were positive in three samples. The immunological techniques have been successfully used in paleoparasitology to detect protozoan infections. Amoebiasis could have been a severe disease in the past, especially where poor sanitary conditions prevailed, and there is evidence that this cemetery may have been used in a situation where poor sanitary conditions may have prevailed. The presence of this protozoan in US during the late XIX century gives information on the health of the population and provides additional data on the parasite's evolution since its appearance in the New World.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Molecular paleoparasitological diagnosis of Ascaris sp. from coprolites: new scenery of ascariasis in pre-Colombian South America times

Paleoparasitological studies using microscopy showed that Ascarisand Trichuris trichiura are the human intestinal parasites most found in archaeological sites. However, in pre-Columbian South American archaeological sites, Ascaris is rare. In this work we standardized a molecular methodology for Ascaris diagnosis directly from ancient DNA retrieved from coprolites. Using cythochrome b gene (142 bp) target, ancient DNA sequences were retrieved from South American samples, negative by microscopy. Moreover, the methodology applied was sensitive enough to detect ancient DNA extracted from 30 Ascaris eggs from an European coprolite. These results revealed a new scenery for the paleodistribution of Ascaris in South America.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.

Intestinal helminthiases in Ecuador: the relatíonship between prevalence, genetic, and socioeconomic factors

Prevalence of infection with the intestinal helminths, Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale and Strongyloides stercoralis was examinedin 632 residents of communities in Esmeraldas province of Ecuador. These communities were divided into two groups according to area of habitation which reflected different socioeconomic circumstances. Attempts were made to correlate infection status with race and ABO blood group phenotype. The racial groups included blacks, Chachi amerindians, and mixed-race mestizos. Greater prevalences of infection were seen in the area oflower socioeconomic status. No racial or blood group associations with helminth infection were seen controlling for socioeconomic status.